Familial Alzheimer's Disease (FAD) is a genetically heritable dominant disorder. Specific missense mutations that result in a single amino acid substitution in p-amyloid precursor protein (APR) have been linked with FAD. FAD mutations in APP cluster around two regions, the extracellular domain adjacent to the a- and (3-secretase sites, and within the intramembranous region adjacent to known y-secretase cleavage sites. The goal of this proposal is to study the effects of FAD mutations upon y-secretase mediated cleavage of APP in the brains of intact mice. The approach involves the development of a trarisgenic mouse model employing a y-secretase activity reporter system previously developed and validated in vitro. Using this screen in cell culture systems, we have demonstrated that FAD mutations result in a decrement in liberation of the APP carboxy-terminus resulting from APP cleavage. While this data is compelling, it remains to be tested whether decrements in y-secretase-mediated APP cleavage are a common effect of FAD mutations within intact brain. This project will test the hypothesis that FAD mutations decrease y-secretase- mediated proteolytic liberation of the carboxy-terminal fragment of APP from the membrane in brain under physiological conditions. The Specific Aims are: 1) Develop a transgenic reporter mouse model to assay y-secretase activity in vivo. Transgenic mouse lines will be developed expressing the APP-Ga!4VP16 activator under the control of the elongation factor 1 alpha (EF1a) promoter, and crossed with Gal4- luciferaseEGFP transgenic mice. Successful deployment of the genetic screening system, and the y-secretase dependency of the reporter output, will be validated by histological and reporter assay methods. 2) Compare y-secretase-mediated cleavage of wild-type and FAD mutant APP-Gal4VP16 using the transgenic reporter mouse model. FAD mutant APP-Gal4VP16 transgenic lines will be crossed with Gal4- luciferaseEGFP reporter mice, and comparative analysis of the reporter output of the wild-type and FAD mutants will be used to determine relative y-secretase activity in each set of transgenic: lines. [unreadable] [unreadable]